Project Summary/Abstract Neurons are terminally post-mitotic cells that use their microtubule arrays not for cell division but rather as architectural elements required for the elaboration of elongated axons and dendrites. In addition to acting as compression-bearing struts that provide for the shape of the neuron, microtubules also act as directional railways for organelle transport. Microtubules in the axon are nearly uniformly oriented, with the plus ends of the microtubules directed away from the cell body (?plus-ends-out?). Preservation of this microtubule pattern is crucial for the normal functioning of an axon throughout the life of the neuron. This microtubule polarity pattern is also important for distinguishing features of the axon from the dendrite, as dendrites of vertebrate neurons have a mixed pattern of microtubule polarity orientation. Long-standing questions in cellular neuroscience are how the plus-end-out polarity pattern of microtubules arises in the axon, how axons maintain this pattern during the life of the neuron and how flaws arising from plastic events in the life of the neuron are repaired. This competing renewal builds on a mechanism called ?polarity sorting,? in which microtubules are organized according to their polarity orientation via their transport by molecular motor proteins. Specifically, the investigators hypothesize that in the case of the healthy axon, microtubules are transported with plus-ends leading, so that plus-end-out microtubules move forward down the axon while minus-end-out microtubules are transported back to the cell body to clear them from the axon. If not for this clearing mechanism, minus-end- out microtubules would accumulate in the axon and corrupt its polarity pattern. The proposed experiments will first test the polarity-sorting hypothesis. Experiments will then be conducted to test the hypothesis that two minus-end-directed molecular motor proteins, namely cytoplasmic dynein and KIFC1, share the responsibility of polarity sorting microtubules in the axon. Finally, studies will be conducted to identify the structures against which the molecular motors generate forces to transport microtubules in the axon. Mobile microtubules are generally quite short, and are hypothesized to move against either long microtubules or actin bundles, depending on the particular motor protein. Computational modeling will add additional rigor to the project, especially in terms of explaining why the axon?s microtubule polarity pattern is corrupted when various molecular players are manipulated. The experiments will utilize contemporary live-cell imaging techniques with unprecedented documentation of the polarity orientation of mobile microtubules in the axon, together with methods for acutely inhibiting molecular motor proteins. The work has relevance to potential treatments for diseases of the nervous system that may corrupt the microtubule polarity pattern of the axon.